Metabolome analysis revealed the critical role of betaine for arsenobetaine biosynthesis in the marine medaka (Oryzias melastigma).

Arsenobetaine (AsB), a non-toxic arsenic (As) compound found in marine fish, structurally resembles betaine (GB), a common methyl donor in organisms. This study investigates the potential role of GB in AsB synthesis in marine medaka (Oryzias melastigma) using metabolomic analysis. Dietary exposure to arsenate (As(V)) and varying GB concentrations (0.05% and 0.1% in diets) increased total As and AsB bioaccumulation, particularly in marine medaka muscle. Metabolomic analysis revealed that GB played a crucial role in promoting up-regulation in methylthioadenosine (MTA) by modulating the methionine cycle and down-regulation in glutathione (GSH) by modulating the glutathione cycle. Methionine metabolism and GSH, potentially binding again to exogenous GB, could synchronously produce more non-toxic AsB. Combining verification experiments of differential metabolites of Escherichia coli in vitro, GB, GSH, S-adenosylmethionine (SAM), and arsenocholine (AsC) entered methionine and glutathione metabolism pathways to generate more AsB. These findings underscore the GB's crucial regulatory role in modulating the synthesis of AsB. This study provides vital insights into the interplay between the structural analogs GB and AsB, offering specific strategies to enhance the detoxification mechanisms of marine fish in As-contaminated environments.

Single-Cell RNA Sequencing Profiling Cellular Heterogeneity and Specific Responses of Fish Gills to Microplastics and Nanoplastics.

Fish gills are highly sensitive organs for microplastic (MP) and nanoplastic (NP) invasions, but the cellular heterogeneity of fish gills to MPs and NPs remains largely unknown. We employed single-cell RNA sequencing to investigate the responses of individual cell populations in tilapia Oreochromis niloticus gills to MP and NP exposure at an environmentally relevant concentration. Based on the detected differentially expressed gene (DEG) numbers, the most affected immune cells by MP exposure were macrophages, while the stimulus of NPs primarily targeted T cells. In response to MPs and NPs, H+-ATPase-rich cells exhibited distinct changes as compared with Na+/K+-ATPase-rich cells and pavement cells. Fibroblasts were identified as a potential sensitive cell-type biomarker for MP interaction with O. niloticus gills, as evidenced by the largely reduced cell counts and the mostly detected DEGs among the 12 identified cell populations. The most MP-sensitive fibroblast subpopulation in O. niloticus gills was lipofibroblasts. Cell-cell communications between fibroblasts and H+-ATPase-rich cells, neurons, macrophages, neuroepithelial cells, and Na+/K+-ATPase-rich cells in O. niloticus gills were significantly inhibited by MP exposure. Collectively, our study demonstrated the cellular heterogeneity of O. niloticus gills to MPs and NPs and provided sensitive markers for their toxicological mechanisms at single-cell resolution.

Novel Near-Infrared-II In Vivo Visualization Revealed Rapid Calcium Intestine Turnover in Daphnia magna with Delayed Impact by Cadmium and Acidification.

Calcium is a highly demanded metal, and its transport across the intestine of Daphnia magna remains a significant unresolved question. Due to technical constraints, the visualization of the kinetic process of Ca passage through D. magna has been challenging. Here, we developed the second near-infrared Ca sensor (NIR-II Ca) and conducted real-time in vivo imaging of Ca in daphnids with a high signal-to-noise ratio, deep tissue penetration, and minimal damage. Through the utilization of the NIR-II Ca sensor, we for the first time visualized and quantified the kinetic process of Ca passage in the intestine in real time. The results revealed that trophically available Ca passed through the intestines in 24 h, whereas waterborne Ca required only 35 min. This rapid "flushing through" mechanism established waterborne Ca as the primary source of Ca absorption. However, environmental stressors such as water acidification and cadmium significantly delayed the Ca passage and absorption. The development of NIR imaging and sensors allows for real-time dynamic visualization of contaminants/nutrients in organisms and holds great potential as a powerful tool for future studies into material kinetic processes in living animals.

Responses of zebrafish (Danio rerio) cells to antibiotic erythromycin stress at the subcellular levels.

Erythromycin (ERY) is one of the most used antibiotics frequently detected in different aquatic environments and may bring burdens to aquatic ecosystems. However, the impacts of antibiotics on aquatic systems other than the antibiotic resistance genes remain largely unknown. In the present study, the responses to ERY exposure at the subcellular-organelle levels were for the first time investigated and imaged over 24 h. Exposure to ERY hampered the zebrafish (Danio rerio) cell growth and decreased the cell viability in a time-dependent mode. Meanwhile, exposure to a low concentration of ERY (73.4 µg L-1) induced reactive oxygen species (ROS) overproduction and lysosomal damage following lysosomal alkalization and swelling. In turn, the lysosomal stress was the major driver of altering the ROS level, superoxide dismutase (SOD) activity, and glutathione (GSH) content. Subsequently, mitochondria displayed dysfunction such as increased mitochondrial ROS, impaired mitophagy, and induced mitochondria-driven apoptosis, as well as impaired mitochondrial electron transport chain and loss of membrane potential. These results collectively demonstrated the subcellular sensitive machinery responses to ERY stress at environmentally relevant and slightly higher sub-lethal concentrations. ERY may induce switching from autophagy to apoptosis with corresponding changes in lysosomal activity, antioxidant activity, and mitochondrial activity. The findings provided important information on the physiological and subcellular responses of fish cells to ERY.

Cell-Type-Dependent Dissolution of CuO Nanoparticles and Efflux of Cu Ions following Cellular Internalization.

CuO nanoparticles (NPs) show promising applications in biosensors, waste treatment, and energy materials, but the growing manufacture of CuO NPs also leads to the concerns for their potential environmental and health risks. However, the cellular fates of CuO NPs such as Cu ion dissolution, transformation, and efflux remain largely speculative. In the present study, we for the first time combined the gold-core labeling and Cu ion bioimaging technologies to reveal the intracellular fates of CuO NPs in different cells following cellular internalization of NPs. We demonstrated that the dissolution rate of CuO NPs depended on the cell type. Following CuO dissolution, limited transformation of Cu(II) to Cu(I) occurred within the cellular microenvironment. Instead, Cu(II) was rapidly eliminated from the cells, and such rapid efflux in different cells was highly dependent on the GSH-mediated pathway and lysosome exocytosis. The labile Cu(I) level in the two cancerous cell lines was immediately regulated upon Cu exposure, which explained their tolerance to Au@CuO NPs. Overall, our study demonstrated a very rapid turnover of Cu in the cells following CuO internalization, which subsequently determined the cellular toxicity of CuO. The results will have important implications for assessing the health risk of CuO NPs.

A green slurry electrolysis to recover valuable metals from waste printed circuit board (WPCB) in recyclable pH-neutral ethylene glycol.

The continuous growth of e-waste necessitates an efficient method to recover their metal contents to improve their recycling rate. The successful recovery of the metallic component from Waste Electrical and Electronic Equipment (WEEE) can generate great economic benefits to incentivize the industrial recycling effort. In this study, we report the use of slurry electrolysis (SE) in pH-neutral ethylene glycol (EG) electrolyte to extract and recover the metallic component from waste printed circuit broad (WPCB) powder. The system operates at room temperature and atmospheric pressure, and the electrolyte can be recycled multiple times with no signs of chemical degradation. The EG electrolyte system can oxidize the metallic component without triggering anodic gas evolution, which allowed us to incorporate a reticulated vitreous carbon (RVC) foam anode to maximize the capture and oxidation of the metal content. The system demonstrated up to 99.1% Faraday efficiency for the cathodic metal deposition and could recover Cu from the WPCB powder in a selective manner of 59.7% in the presence of 12 other metals. The SE reaction system was also scalable and displayed no compromises on the Cu recovery selectivity. With the ability to leach and recover metallic content from WPCB in a mild and chemically benign condition, the SE system displayed much promise to be adapted for industrial-scale metal recovery from WPCB.

Functional heterogeneity of immune defenses in molluscan oysters Crassostrea hongkongensis revealed by high-throughput single-cell transcriptome.

Oyster is the worldwide aquaculture molluscan and evolves a complex immune defense system, with hemocytes as the major immune system for its host defense. However, the functional heterogeneity of hemocyte has not been characterized, which markedly hinders our understanding of its defense role. Here, we used the single-cell transcriptome profiling (scRNA-seq), which provides a high-resolution visual insight into its dynamics, to map the hemocyte and assess its heterogeneity in a molluscan oyster Crassostrea hongkongensis. By combining with the cell type specific RNA-seq, thirteen subpopulations belonging to granulocyte, semi-granulocyte, and hyalinocyte were revealed. The granulocytes mainly participated in immune response and autophagy process. Pseudo-temporal ordering of granulocytes identified two different cell-lineages. The hematopoietic transcription factors regulated networks controlling their differentiations were also identified. We further identified one subpopulation of granulocytes in immune activate states with the cell cycle and immune responsive genes expressions, which illustrated the functional heterogeneity of the same cell type. Collectively, our scRNA-seq analysis demonstrated the hemocytes diversity of molluscans. The results are important in our understanding of the immune defense evolution and functional differentiation of hemocytes in Phylum Mollusca.

Methylmercury biomagnification in aquatic food webs of Poyang Lake, China: Insights from amino acid signatures.

As the dominant mercury species in fish, methylmercury (MeHg) biomagnifies during its trophic transfer through aquatic food webs. MeHg is known to bind to cysteine, forming the complex of MeHg-cysteine. However, relationship between MeHg and cysteine in large-scale food webs has not been explored and contrasted with MeHg biomagnification models. Here, we quantified the compound-specific nitrogen isotopic analysis of amino acids (CSIA-AA), MeHg, and amino acid composition in aquatic organisms of Poyang Lake, the largest freshwater lake in China. The trophic positions (TPAA) of organisms ranged from 1.0 ± 0.1-3.7 ± 0.2 based on CSIA-AA approach. The trophic magnification factor (TMF) of MeHg, derived from the regression slope of Log-transformed MeHg in organisms upon their TPAA for the entire food web was 9.5 ± 0.5. Significantly positive regression between MeHg and cysteine (R2 = 0.64, p < 0.01) was documented, suggesting MeHg-cysteine complex may potentially play a critical role in the bioaccumulation of MeHg. Furthermore, TMFs of MeHg calculated with and without cysteine normalization compared well (7.7-8.7) when excluding primary producers. Our results implied that MeHg may biomagnify as the complex of MeHg-cysteine and contribute to our understanding of MeHg trophic transfer at the molecular level.

New insights into the chemical forms of extremely high methylmercury in songbird feathers from a contaminated site.

The chemical forms of mercury (Hg), particularly methylmercury (MeHg), in songbird feathers from an abandoned mining region were analyzed via X-ray absorption near-edge structure analysis (XANES). In feathers, proportions of MeHg as total mercury (75.6-100%) quantified by the XANES were directly comparable to the chemical extraction values (74.1-95.9%). Most of MeHg were bound with cysteine (Cys) and reduced glutathione (GSH), whereas inorganic mercury (IHg) was mainly bound with GSH. These results were consistent with those found in fish muscles and human hairs of both fish consumers and occupational Hg exposure populations. Our study suggested that chemical forms and speciation of Hg were highly dependent on the exposure sources and food consumption, respectively. Bird feathers were able to selectively accumulate MeHg due to their special binding ways. However, detailed mechanisms of Hg accumulation in bird feathers remain to be further elucidated.

Dominant Role of Silver Ions in Silver Nanoparticle Toxicity to a Unicellular Alga: Evidence from Luminogen Imaging.

Despite the extensive studies on silver nanoparticle (AgNP) toxicity, the contribution of released silver ions to the toxicity still remains debatable. This study investigated the toxicity of AgNPs with different sizes (20 and 60 nm) toward a phytoplankton Euglena gracilis, with a focus on evaluating the contributions of dissolved Ag to the toxicity by combining a newly developed aggregation-induced emission luminogen (AIEgen)-based imaging technique and a traditional inductively coupled plasma mass spectrometry (ICP-MS) method. Smaller AgNPs, which dissolved much faster, exhibited greater toxicity, as evidenced by lower 50% growth inhibition concentration (EC50). However, the average Ag+ concentration at each EC50 was comparable to the EC50 of AgNO3, and similar subcellular Ag distribution patterns were observed in both AgNPs and AgNO3 exposed algae. More silver ions were internalized in algae treated with smaller AgNPs. With the application of luminogen imaging technology, we for the first time demonstrated that few Ag(I) were presented intracellularly in algae exposed to AgNPs when any released Ag in the medium was complexed by cysteine. Ag(I) was mainly distributed in cellular debris, organelles, and metal-rich granules fractions. No conclusive evidence for AgNP internalization was documented. Our results strongly suggested that Ag+ released from AgNPs extracellularly dominated the AgNP toxicity. The findings of this work provide new and useful insight into the toxicity of AgNPs in aquatic environments.

Uniquely high turnover of nickel in contaminated oysters Crassostrea hongkongensis: Biokinetics and subcellular distribution.

Despite the environmental concerns regarding nickel (Ni) especially in China, it has received little attention in aquatic animals due to its comparatively weak toxicity. In the present study, we explored the bioaccumulation, biokinetics, and subcellular distribution of Ni in an estuarine oyster Crassostrea hongkongensis. We demonstrated that Ni represented a new pattern of bioaccumulation in oysters characterized by rapid elimination and low dissolved uptake. The waterborne uptake rate constant and dietary assimilation efficiency were 0.036L/g/h and 28%, respectively, and dissolved uptake was the predominant exposure route. The efflux rate constant was positively related to tissue Ni concentration, with the highest efflux of 0.155d-1. Such high elimination resulted in a high Ni turnover and steady-state condition reached rapidly, as shown with a 4-week waterborne exposure experiment at different Ni concentrations. Ni in oysters was mainly sequestered in metallothionein-like protein (MTLP), metal-rich granule, and cellular debris. MTLP was the most important binding fraction during accumulation and depuration, and played a dynamic role leading to rapid Ni elimination. Pre-exposure to Ni significantly reduced the dissolved uptake, probably accompanied by depressed filtration activity. Overall, the high turnover and regulation of Ni in oysters were achieved by enhanced efflux, suppressed uptake, and sequestration of most Ni into the detoxified pool.

Chronic effects of copper in oysters Crassostrea hongkongensis under different exposure regimes as shown by NMR-based metabolomics.

Traditional metal toxicity tests on organisms have mainly focused on continuous exposure at a fixed concentration. However, organisms are more likely exposed to pollutants intermittently in estuarine environments that are significantly impacted by anthropogenic activity. The present study examined whether different copper (Cu) exposure regimes at an equivalent dose can induce different metabolomics effects on the oysters. An estuarine oyster Crassostrea hongkongensis was exposed to Cu continuously or intermittently at an equal dose (time × concentration) for 6 wk. Continuous exposure regimes included 2 doses of 3.3 µg/L for 24 h and 20 µg/L for 24 h, with corresponding equal doses of 2 intermittent exposure regimes of 20 µg/L for 4 h and 120 µg/L for 4 h, respectively. Time-course measurements suggested that Cu bioaccumulation was comparable at equal low doses between the continuous regime (3.3 µg/L for 24 h) and the intermittent regime (20 µg/L for 4 h), but there was considerable difference for the high dose under different regimes. Nuclear magnetic resonance (NMR)-based metabolomics suggested that continuous and intermittent Cu exposures led to similar metabolite variation pattern in gills at an equal high dose, including decreased amino acids (e.g., aspartate, glycine, isoleucine, leucine, lysine, phenylalanine, threonine, and valine), lower energy-related compounds (e.g., adenosine triphosphate/adenosine diphosphate, acetate, citrate, and glycogen), and altered osmolytes (e.g., homarine and taurine). These biomarkers indicated disturbance of osmotic regulation and energy metabolism induced by Cu exposure regardless of regime. In addition, the 4-h intermittent Cu exposure resulted in slightly fewer adverse effects compared with the corresponding equal-dose continuous exposure. Oysters appeared to recover during the intervals of Cu exposure. The results indicated that metabolomic effects induced by Cu were more dose dependent than the Cu exposure regime. Environ Toxicol Chem 2017;36:2428-2435. © 2017 SETAC.

Novel insights into iron regulation and requirement in marine medaka Oryzias melastigma.

Iron (Fe) is an essential trace element for marine fish. However, our knowledge of Fe requirements at different development stages of marine fish is still limited. Here, we reported the efficient Fe absorption strategies adopted by larval fish under different dietary Fe supplementary levels (i.e., 0-640 mg/kg). Biokinetically, the larval fish controlled their dietary Fe assimilation efficiency (AE, 1.6-18.5%), and enhanced their waterborne Fe uptake (ca. 2.5 fold change of uptake rate constant) once the dietary Fe was deficient (i.e., 27.4 mg Fe/kg feed). Transcriptionally, the expression of hepcidin1 (hep1; Fe regulator; i.e., 2.3-15.7 fold change) in larval fish was positively correlated with the Fe supplementary levels. Comparatively, the female adult fish were poor in assimilating the added Fe source (i.e., ferric form) with similar life-sustainable levels of Fe (i.e., 0.046-0.12 µg/g/d assimilated for Fe supplementary levels of 27.4, 162 and 657 mg Fe/kg feed). The overall feeding experiments suggested that dietary net Fe flux sufficient for the normal growth of larval medaka was 0.71-1.75 µg/g/d (i.e., 83.9 mg Fe/kg feed), consistent with the modeled value (i.e., 1.09-2.16 µg/g/d). In female adults, the estimated essential net Fe flux was 0.88-0.90 µg/g/d.

Reduced cadmium accumulation and toxicity in Daphnia magna under carbon nanotube exposure.

With increasing application and commercial production, carbon nanotubes (CNTs) will inevitably be released into aquatic environments and affect the transport and toxicity of toxic metals in ecosystems. The present study examined how CNTs affected the biokinetics and toxicity of a toxic metal, cadmium (Cd), in the freshwater zooplankton Daphnia magna. The authors quantified the dissolved uptake and the 50% lethal concentration (LC50, 48 h and 72 h) of Cd in daphnids in the presence of functionalized multiwalled nanotubes (F-CNTs) with different lengths (10-30 µm vs 0.5-2 µm) and concentrations (4 mg/L and 8 mg/L). Compared with the control treatment without CNTs, both CNTs slowed down the accumulation rate of Cd in D. magna over 8 h of exposure and further reduced the accumulation thereafter. Mechanisms for the reduced Cd uptake were mainly related to the influences of CNTs on the physiological activity of daphnids. The LC50 of D. magna in the presence of Cd and shorter CNTs was almost the same as that of the control group without CNTs. However, the LC50 of the groups with normal CNTs was significantly higher than that of the control group (i.e., F-CNTs decreased Cd toxicity significantly). Meanwhile, CNTs also decreased the tolerance of D. magna to Cd. The present study suggests that different physical properties of CNTs, such as length, need to be considered in the environmental risk assessment of CNTs.

Low bioavailability of silver nanoparticles presents trophic toxicity to marine medaka (Oryzias melastigma).

Concerns for the potential risks of silver nanoparticles (AgNPs) to aquatic organisms have increased. The present study investigated the trophic transfer of AgNPs from brine shrimp (Artemia salina) nauplii to marine medaka. We found that the aggregated AgNPs (20 and 80 nm) and well dispersed 80-nm AgNPs (stabilized by 20 µM Tween 20) could be readily accumulated by brine shrimp, while far less well-dispersed 20-nm AgNPs were accumulated. The assimilation efficiency (AE) of AgNPs in medaka fed AgNPs-contaminated brine shrimp was low (<6%), resulting in a low trophic transfer efficiency (0.01-0.04) after 28 days of chronic dietary exposure. However, such low bioavailability of dietary AgNPs could inhibit the whole-body Na+/K+-ATPase and superoxide dismutase (SOD) activity in the fish within the first 2 weeks of exposure. Significant (p<0.05, two-way ANOVA) inhibition occurred in the high AgNPs-contaminated brine shrimp treatment over 28 days of chronic exposure. Furthermore, reduced growth and water content percentage were also observed in fish fed high dosages of AgNPs-contaminated brine shrimp. Our study highlighted the potential of trophically available AgNPs in bringing toxicity to the marine fish.

Influences of ambient carbon nanotubes on toxic metals accumulation in Daphnia magna.

With the rapid increase of carbon nanotube (CNT) applications, there are considerable concerns of their inevitable releases into the aquatic environments. CNTs may interact with and further influence the fate and transport of other pollutants such as toxic metals. In the present study, non-covalent and nontoxic dispersant polyvinyl pyrrolidone (PVP) was used to provide a relatively stable test solution for CNTs. The dissolved uptake rate constant (ku) and the dietary assimilation efficiency (AE) of cadmium (Cd) and zinc (Zn) were then quantified in a freshwater zooplankton Daphnia magna in the presence of different CNTs (without functionalized - single-walled nanotubes-SWNTs, multi-walled nanotubes-MWNTs, and with functionalized - F-SWNTs, F-MWNTs, containing oxygen functional groups at the defect sites of CNTs) concentrations. We demonstrated that different CNTs exposures led to distinctive metal accumulation patterns. Non-functionalized CNTs significantly decreased the metal uptake rate from the dissolved phase, possibly because of their effects on the physiological activity of animals. In contrast, the F-CNTs (F-SWNTs and F-MWNTs) adsorbed the metals and increased the metal accumulation in daphnids in a concentration-dependent manner, due to the ingestion of F-CNTs associated metals. The AEs of metals in D. magna were elevated by CNTs physical blocking of the animal guts. Our present study showed that CNTs could serve as a new pathway for metal accumulation. This raised a new environmental problem of CNTs since they may induce the accumulation of toxic metals from the dietary exposure.

Regulation of sodium and calcium in Daphnia magna exposed to silver nanoparticles.

The toxicity of manufactured silver nanoparticles (AgNPs) has been widely studied, but the influence of AgNPs on the major ions (such as sodium [Na] and calcium [Ca]) regulations are unknown. In the present study, a freshwater cladoceran Daphnia magna was exposed to commercial AgNPs coated with polyvinylpyrrolidone. After 48 h, the Na body content was significantly reduced by AgNO3 exposure, but the Ca body content was significantly increased under AgNO3 and AgNP exposures, respectively. No effect was observed on the body concentrations of Na and Ca at 50 to 500 µg/L AgNPs with 1-µM cysteine addition. Exposure of AgNO3 and AgNPs inhibited the Na influx and elevated the Na efflux. In contrast, their exposure increased the Ca influx, but did not affect the Ca efflux. The results of the present study demonstrated the significant influences of AgNO3 and AgNPs (without cysteine) on Na and Ca regulations. Such effect of AgNPs on Na and Ca regulation disappeared after cysteine addition, indicating that the soluble Ag released from AgNPs played a major role in the ionoregulatory dysfunction.

Facilitated bioaccumulation of cadmium and copper in the oyster Crassostrea hongkongensis solely exposed to zinc.

Exposure to one metal might have significant effects on the bioaccumulation of other metals. In the present study, we examined the possible effects of Zn exposure on the bioaccumulation of Cd and Cu in three populations of the oyster Crassostrea hongkongensis. We found that Zn exposure significantly enhanced the tissue concentrations of Cd and Cu in all populations, and the tissue concentrations of Cd and Cu were highly and positively related to the tissue Zn concentration. Furthermore, the enhanced bioaccumulation of Cd and Cu resulted mainly from their increasing accumulation and distribution in two subcellular fractions (i.e., metallothionein-like proteins and metal-rich granules). Tissue concentrations of Cd and Cu in the natural Zn-contaminated oysters also covaried with tissue Zn concentration, and prediction analyses revealed that Zn exposure was a significant contributor to tissue Cd and Cu concentrations. Therefore, we concluded that the increased Zn bioavailability in ambient waters not only increased the tissue Zn concentration but also enhanced the overall bioaccumulation of Cd and Cu. This study strongly demonstrates that contamination of metals in oysters may result from concurrent exposure to other metals. Thus, environmental managers should consider the possible exposure to other metals such as Zn in order to interpret/predict the tissue concentrations of toxic metals in oysters.

Size-dependent uptake of silver nanoparticles in Daphnia magna.

The toxicity of silver nanoparticles (AgNPs) has been widely investigated, but the process of bioaccumulation such as uptake remains less studied. In the present study, we employed the radioisotope ((110m)Ag) to trace the behaviors of commercial AgNPs with three nominal particle sizes (20, 50, and 100 nm) and two surface coatings (citrate and tannic acid) in a model organism Daphnia magna . The size distributions of AgNPs in the medium increased continuously as the exposure time increased, especially for the smallest AgNPs (20 nm). Cysteine, the amino acid containing thiol group, significantly enhanced particle aggregation, with a 30-fold increase of the hydrodynamic size for AgNPs with 20 nm nominal size after 6 h of exposure. We demonstrated that the influx rates of AgNPs into daphnids were size-dependent. At 500 µg/L AgNPs with 1 µM cysteine, the influx rates of AgNPs were in the sequence 20 nm > 50 nm > 100 nm (nominal size) for both types of surface coatings. Such sequence was consistent with the size distribution in the medium. More than 60% of AgNPs were distributed in the gut of daphnids, indicating that ingestion was the dominant uptake pathway. The size-dependent influx rate was also observed at different AgNPs concentrations. The measured uptake rate constant was lower than that of AgNO(3) at low AgNPs concentration, but it became higher at high AgNPs concentrations. Our study highlighted the indispensability of characterizing the size distribution of AgNPs dispersed in the medium in studying the AgNPs uptake. The accurate quantification of AgNPs influx rate suggested an uptake pathway entirely different from that of AgNO(3) in the daphnids.

Two-photon excitation chlorophyll fluorescence lifetime imaging: a rapid and noninvasive method for in vivo assessment of cadmium toxicity in a marine diatom Thalassiosira weissflogii.

We demonstrate that a two-photon excitation fluorescence lifetime imaging technology can rapidly and noninvasively assess the cadmium (Cd)-induced toxic effects in a marine diatom Thalassiosira weissflogii. The chlorophyll, an intrinsic fluorophore, was used as a contrast agent for imaging of cellular structures and for assessment of cell toxicity. The assessment is based on an imaging-guided statistical analysis of chlorophyll fluorescence decay. This novel label-free imaging method is physically based and free of tedious preparation and preprocessing of algal samples. We first studied the chlorophyll fluorescence quenching induced by the infrared two-photon excitation laser and found that the quenching effects on the assessment of Cd toxicity could be well controlled and calibrated. In the toxicity study, chlorophyll fluorescence lifetime images were collected from the diatom samples after exposure to different concentrations of Cd. The alteration of chloroplast structure at higher Cd concentration was clearly identified. The decay of chlorophyll fluorescence extracted from recorded pixels of high signal-to-noise ratio in the fluorescence lifetime image was analyzed. The increase of average chlorophyll fluorescence lifetime following Cd treatment was observed, indicating the Cd inhibition effect on the electron transport chain in photosynthesis system. The findings of this study show that the temporal characteristics of chlorophyll fluorescence can potentially be utilized as a biomarker for indicating Cd toxicity noninvasively in algal cells.